1. Technical Field
The invention relates to IEX-1 knockout animals and methods and materials related to IEX-1 knockout animals, hypertension, and vascular smooth muscle cells.
2. Background Information
IEX-1 (also known as hdif and PPRG in humans) is an immediate early gene in humans and is known as gly96 in the mouse (Kumar et al., 1998. A novel immediate early response gene, IEX-1, is induced by ultraviolet radiation in human keratinocytes. Biochem Biophys Res Commun 253:336–341; Kondratyev et al., 1996. Identification and characterization of a radiation-inducible glycosylated human early-response gene. Cancer Res 56:1498–1502; Schafer et al., 1999. Human PACAP response gene 1 (p22/PRG1): proliferation-associated expression in pancreatic carcinoma cells. Pancreas 18:378–384; Schafer et al., 1996. PRG1: a novel early-response gene transcriptionally induced by pituitary adenylate cyclase activating polypeptide in a pancreatic carcinoma cell line. Cancer Res 56:2641–2648; Pietzsch et al., 1998. Genomic organization, promoter cloning, and chromosomal localization of the Dif-2 gene. Biochem Biophys Res Commun 245:651–657; Charles et al., 1993. Genomic structure, cDNA sequence, and expression of gly96, a growth factor-inducible immediate-early gene encoding a short-lived glycosylated protein. Oncogene 8:797–801). The IEX-1 polypeptide was identified in endothelial and vascular smooth muscle cells (VSMCs; Kumar et al., 1998. A novel immediate early response gene, IEX-1, is induced by ultraviolet radiation in human keratinocytes. Biochem Biophys Res Commun 253:336–341; Feldmann et al., 2001. Expression of an immediate early gene, IEX-1, in human tissues. Histochem Cell Biol 115:489–497). IEX-1 is highly expressed in the endothelium and VSMCs of both arteries and veins. Recent information shows that IEX-1 is induced by biomechanical strain in cardiomyocytes, VSMCs, and monocytes, and that it inhibits cardiomyocyte hypertrophy and VSMC and the response to vascular injury (Lehoux and Tedgui, 2003. All strain, no gain: stretch keeps proliferation at bay via the NF-kappaB response gene iex-1. Circ Res 93:1139–1141; Schulze et al., 2003. Biomechanically induced gene iex-1 inhibits vascular smooth muscle cell proliferation and neointima formation. Circ Res 93:1210–1217; Ohki et al., 2002. Identification of mechanically induced genes in human monocytic cells by DNA microarrays. J Hypertens 20:685–691; and De Keulenaer et al., 2002. Identification of IEX-1 as a biomechanically controlled nuclear factor-(kappa)B target gene that inhibits cardiomyoctye hypertrophy. Circulation Research 90:685–691).
IEX-1 polypeptides are cytoplasmic and nuclear glycosylated proteins. The IEX-1 gene encodes a 156 amino acid protein with a calculated MW=16927.48 Da and an estimated pI=8.83. The protein has various sites at which post-translational modifications such as phosphorylation can potentially occur that may be mediated by cAMP-dependent kinase, protein kinase C, and various phosphatases. In addition, a glycosylation site is observed at amino acid residues 133–135. A nuclear localization signal, RKRSRR, and a co-activator motif, LXXL, are also present. On gels, the in vitro translated protein has a Mr of ˜17–20,000 Da and upon glycosylation with pancreatic microsomal membranes the protein has a Mr of 27,000 Da. Western blot analysis of tissue IEX-1 shows a protein with a Mr of 27,000 on SDS-PAGE, suggesting that the protein is post-translationally modified in cells in vivo.